Research Highlights

A high-throughput chemically induced inflammation assay in zebrafish

ABSTRACT:

Background: Studies on innate immunity have benefited from the introduction of zebrafish as a model system. Transgenic fish expressing fluorescent proteins in leukocyte populations allow direct, quantitative visualization of an inflammatory response in vivo. It has been proposed that this animal model can be used for high-throughput screens aimed at the identification of novel immuno-modulatory lead compounds. However, current assays require invasive manipulation of fish individually, thus preventing high content screening.

Results: Here, we show that specific, non-invasive damage to lateral line neuromast cells can induce a robust acute inflammatory response. Exposure of fish larvae to sub-lethal concentrations of copper sulfate selectively damages the sensory hair cell population inducing infiltration of leukocytes to neuromasts within 20 minutes. Inflammation can be assayed in real-time using transgenic fish expressing fluorescent proteins in leukocytes or by histochemical assays in fixed larvae. We demonstrate the usefulness of this method for chemical and genetic screens to detect the effect of immuno-modulatory compounds and mutations affecting the leukocyte response. Moreover, we transformed the assay into a high-throughput screening method by using a customized automated imaging and processing system that quantifies the magnitude of the inflammatory reaction.

Conclusions: This approach allows rapid screening of thousands of compounds or mutagenized zebrafish for effects on inflammation and enables the identification of novel players in the regulation of innate immunity and potential lead compounds towards new immuno-modulatory therapies. We have called this method the Chemically-Induced Inflammation Assay, or ChIn Assay. See Commentary article: http://www.biomedcentral.com/1741-7007/8/148.

Abstract: Zebrafish embryos offer a unique combination of high-throughput capabilities and the complexity of the vertebrate animal for a variety of phenotypic screening applications. However, there is a need for automation of imaging technologies to exploit the potential of the transparent embryo. Here we report a high-throughput pipeline for registering domain-specific reporter expression in zebrafish embryos with the aim of mapping the interactions between cis-regulatory modules and core promoters. Automated microscopy coupled with custom-built embryo detection and segmentation software allowed the spatial registration of reporter activity for 202 enhancer-promoter combinations, based on images of thousands of embryos.

The diversity of promoter-enhancer interaction specificities underscores the importance of the core promoter sequence in cis-regulatory interactions and provides a promoter resource for transgenic reporter studies. The technology described here is also suitable for the spatial analysis of fluorescence readouts in genetic, pharmaceutical or toxicological screens.

A toxicogenomic barcode

Changes in gene expression patterns in zebrafish embryos resulting from exposure to environmental toxins can identify the individual toxins at work, according to research published by Yang et al. in the online open access journal Genome Biology [ PubMed ]. The genetic response of zebrafish to each toxin can be read like a barcode, offering a potential method for identifying the effects of the toxin on developing vertebrate embryos. Zebrafish embryos were exposed to eleven common pollutants, including cadmium, mercury, dioxin and DDT. Zebrafish have previously found a role in toxicology tests, for example in testing sewage for the presence of toxins. However, these previous tests involved looking at adult fish or embryos. The new method does distinguish individual genetic barcodes of chemicals and will help cope with the high demand from regulators and industry for reliable methods needed to evaluate the developmental toxicity of pharmaceuticals, industrial chemicals and waste products.

Development is controlled by synthesis and degradation

The development of the vertebrate embryo follows a series of hierarchical decisions that eventually lead to the expression of different messenger RNAs (mRNA) in the many different cell types of the adult body. It was until now believed that development of the embryo is predominantly controlled by transcriptional activation of genes. In their recent paper, Ferg and colleagues provide evidence that mRNA degradation plays also an important role. They discovered a molecular mechanism that links maternal mRNA degradation to the transcriptional activation of the zygotic genome, a key process in the commencement of differentiation of the vertebrate embryo. Ferg and colleagues demonstrated that the TATA binding protein, which was once considered a general component of transcription initiation has a specific function in activating only a subset of genes during the blastula stage of the embryo. They showed that TBP is required for the function of a small interfering RNA miR-430 that mediates mRNA degradation. These findings may have also important implications for the activity of embryonic stem cells as activation of the zygotic genome is accompanied by the first cell differentiations and loss of the totipotency of the cells.

The paper appeared in the EMBO J (vol. 26, 3945-56) [ PubMed ] and is featured with a news report in EMBO Reports.

Splicing Segregation: Out of the Nucleus into Cell Proliferation

The functional relevance and the evolution of the two parallel mRNA splicing systems in eukaryotes - a major and a minor spliceosome - are poorly understood. In their recent publication, König and colleagues show an essential and conserved role for the minor spliceosome in cell proliferation and found that it acts in the cytoplasm, thus separated from its major counterpart in the nucleus. The unexpected subcellular segregation of the splicing machines appears to be a highly conserved property, and may account for the evolution of the two independent systems. In addition, cytoplasmic splicing can provide a basic means to regulate pre-mRNA processing and gene expression throughout the cell cycle.